1. Field of the Invention
The present invention relates, in general, to an isolated or purified Ketogulonigenium plasmid endogenous to microorganism strain NRRL B-30035 (ADM 291-19).
2. Background Information
The exploitation of microorganisms to synthesize vitamin C or its chemical pathway intermediates has both economic and ecological advantages. One key intermediate in vitamin C synthesis is 2-keto-L-gulonic acid (2-KLG), which is easily converted chemically to L-ascorbic acid (vitamin C) by esterification followed by lactonization (Delic, V. et al., xe2x80x9cMicrobial reactions for the synthesis of vitamin C (L-ascorbic acid,xe2x80x9d in Biotechnology of Vitamins, Pigments and Growth Factors, Vandamme, E. J., ed., Elsevier Applied Science (London and New York) pp. 299-336 (1989)). Members of a number of bacterial genera have been identified that produce 2-KLG from the oxidation of L-sorbose. Such 2-KLG producing genera include the acidogenic, alpha-proteobacteria Gluconobacter and Acetobacter, the gamma-proteobacteria Pseudomonas, Escherichia, Klebsiella, Serratia and Xanthmonas, the Gram positive Bacillus, Micrococcus, and the unofficial genus Pseudogluconobacter (Imai, K. etal., U.S. Pat. No. 4,933,289 (1990), Sugisawa, H. et al., xe2x80x9cMicrobial production of 2-keto-L-gulonic acid from L-sorbose and D-sorbitol by Gluconobacter melanogenus,xe2x80x9d Agric. Biol. Chem. 54:1201-1209 (1990), Yin, G. et al., U.S. Pat. No. 4,935,359 (1990) and Nogami, I. et al., U.S. Pat. No. 5,474,924 (1995)).
To aid in increasing the yield of bacterial products, attempts have been made to exploit endogenous plasmids within microorganism strains. (Beppu, T. et al., U.S. Pat. No. 5,580,782 (1996), Fujiwara, A. et al., U.S. Pat. No. 5,399,496 (1995)).
One aspect of the invention provides an isolated or purified nucleic acid molecule comprising a polynucleotide having a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of: a nucleotide sequence in SEQ ID NO: 1; a nucleotide sequence of an endogenous plasmid contained in NRRL Deposit No. B-30035; and a nucleotide sequence complementary to any of the above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 97%, 98% or 99% identical, to any of the above nucleotide sequences, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide having a nucleotide sequence as in the above. The polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
Further advantages of the present invention will be clear from the description that follows.